A number of sectors of technology require greater exploration of both chemical and biological space and diversity. In the pharmaceutical sector, for example, the search for new drug candidate compounds is leading to compounds being made and screened at an ever increasing rate. The resulting increase in demand for chemical and biological information is driving innovations in the handling and detection of small volumes of liquids in microfluidic devices.
Microfluidics is a technology that allows the storage, dosing, movement and mixing of very small volumes of fluids and can be applied at the system, module and component level. The technology employs microfluidic components which may be either passive or active. Typically, passive components comprise miniaturised plates that may be functionalised, whilst active components are capable of performing either a unique function, such as sample preparation, or integrated functions, which could incorporate the combined operations of sample preparation, separation and detection.
Both active and passive components may comprise one or more channels which typically have dimensions in the range of micrometres to sub-millimetre, thereby yielding minimum handled volumes in the pL range. The fluidic architecture is formed in the substrate using a variety of microfabrication processes including etching, injection moulding, embossing, laser ablation and stamping. Fluid samples are typically contained and transported within these channels. Common fluids used in microfluidic devices includes whole blood samples and bacterial suspensions. Other features that can be formed in the substrate to contain the sample include well structures. Such microfluidic devices will be familiar to those of ordinary skill in the art.
Miniaturised plates employ a variety of substrates including glass or polymer and may be either planar or may comprise micro-well plates. Planar substrates that are functionalised with bioactive components are commonly known as biochips, and are used for monitoring ligand receptor binding. Micro-well plates, on the other hand, comprise an array of individual wells in a two-dimensional format. Increasing the density of micro-well plates provides some advantages in evaporation losses of the liquid, but also leads to increasing complexity in operations such as dispensing and detection. The dispensing of reagents into micro-well plates is generally performed using robotic systems.
Active microfluidic components which are required to perform a variety of integrated functions, such as sample preparation, separation and detection, require highly sophisticated manipulation of very small volumes of liquids. Sample separation may be performed by any of a variety of well known techniques including, for example, gel electrophoresis and chromatography. These active devices would be familiar to those of ordinary skill in the art, and are alternatively referred to as Lab-on-a-chip or μTAS. Such devices have a wide range of applications, including chemical and biological analysis, and high throughput screening, in addition to microreactor applications.
Many techniques of detection have been implemented in microfluidic devices, including spectroscopic and electrochemical detection. Specifically, absorbance methods can be applied by measuring across either the channel length or the channel width of a microfluidic device but, in view of the short path length in microfluidic devices, such techniques have the disadvantage of relatively low sensitivity in comparison to macroscopic devices.
Optical cavity methods are becoming more widely used as sensitive methods of absorbance measurements. These methods rely on light being confined between two highly reflective mirrors, thereby resulting in the base path length being increased by many orders of magnitude in the gas phase. The first implementation of this technique was Cavity Ring Down Spectroscopy (CRDS), which was proposed by O'Keefe and Deacon1 in 1988. Typically, light from a pulsed laser or a continuous wave (CW) laser with a suitable interruption method is introduced into the cavity through the back of one of the mirrors. The 1/e decay time, known as the ring down time, of a pulse of laser light confined between the mirrors is then measured in the presence and absence of the sample and related to the absorption coefficient at a particular wavelength of the sample in the optical cavity. The wavelength can be scanned in most cases to record an absorption spectrum. However, the detection of the light exiting the cavity requires fast response detectors and associated equipment capable of measuring on the nanosecond timescale and this consideration, along with the expense of pulsed laser sources, makes most implementations of CRDS prohibitively expensive and inconvenient.
Subsequently, Englen et al2 and O'Keefe3 disclosed simpler variations of CRDS which they respectively named Cavity Enhanced Absorption Spectroscopy (CEAS) and Integrated Cavity Output Spectroscopy (ICOS) although, in essence, these techniques are equivalent. In the case of CEAS, a continuous wave light source is used which replenishes the light lost due to reflection inefficiencies and absorption by the sample. Englen et al have shown that light within the cavity reaches steady state within a few ring down times, and its intensity is proportional to the ring down time. Consequently, the sample absorbance can be determined by steady state intensity measurement in the presence and absence of the sample in the cavity and this means that slower response detectors can be used, thereby reducing the cost of the detection element of the experimental scheme. Unfortunately, however, a further consequence is that the absorption cross section now cannot be measured directly and, instead, a comparison with a reference compound in the cavity is firstly required.
Recently, simpler and cheaper light sources have also been proposed for such applications4,5. These include broadband light sources such as arc lamps or high intensity LEDs, both coupled with multiplex detection, thereby in principle allowing the measurement of the entire absorption spectrum in one action, rather than requiring scanning across the spectrum. There is an associated disadvantage, however, which is typically manifested as a lower wavelength resolution for the absorption spectrum.
The CRDS and CEAS techniques have principally been used for the detection of gases which have narrow absorption features. More recently, however, these techniques have been used for the analysis of liquids in which most absorption features are relatively broad (several nanometers linewidth). Thus, Zare6-8 has demonstrated the analysis of liquid samples using CRDS with an inexpensive diode laser source, whilst Ariese9 has described CRDS in the liquid phase for Liquid Chromatography (LC) analysis using a cell made from a silicone rubber spacer clamped leak tight between two high reflectivity mirrors. The mirrors are in direct contact with the liquid flow.
Several prior art documents are available which disclose different multipass techniques, designed to enhance the path length of measurement. Thus, US-A-2005/0162652 teaches doubling the pathlength through microlitre sized liquid samples using LEDs together with a novel implementation of corner cube beamsplitters. However, the maximum enhancement over a conventional absorption measurement is a factor of two.
GB-A-2284904 is concerned with the use of a liquid core fibre optic as a waveguide to achieve long pathlengths in a liquid analyte by choosing a material for the construction of the waveguide which has a lower refractive index than the liquid core. Thus, light from a suitable light source passes along the waveguide through total internal reflection, such that the path length can, in principle, be doubled by using a mirror at one end of the fibre optic to reflect the light back.
U.S. Pat. No. B6,224,830 relates to the improvement of the sensitivity of absorption measurements in microfluidic devices by increasing the pathlength across a microchannel through depositing mirrors on opposite sides of the channel and using the channel as a waveguide to allow multiple reflections from the input and output end of the radiation source. Thus, the light source enters the waveguide and is reflected off the mirrors several times along the length of the fluidic channel as it progresses from the entrance to the exit port.
US-A-2005/0046851 discloses the doubling of the pathlength through a miniature gas cell using folded optics, the process involving depositing mirrors onto the surface of the gas cell. In this approach, the light source enters the gas cell and is reflected off the deposited mirror and onto the detector. Thus, the enhancement achieved using this approach is simply a doubling of the pathlength. Furthermore, the disclosed method is only relevant for measurements relating to gaseous systems.
U.S. Pat. No. B6,839,140 describes the application of cavity enhanced absorption spectroscopy (CEAS) to liquids. Thus, external mirrors of 2-4 mm diameter with typical mirror separations of 0.1-2 mm are assembled into a flow cell giving a minimum interrogated sample volume of 0.5 μL. Clearly, it would be desirable to provide systems with mirror separations of much smaller magnitude.
However, each of these approaches only allows the fluid to pass through the optical cavity, and there is no potential for the fluid to interact with a functionalised surface, or for the performance of complex micro and nanoscale volume fluidic operations, such as the mixing, direction and separation of reagent and sample streams in an integrated approach. Therefore, the existing techniques would be difficult to miniaturise for application to very small scale situations and also would not readily facilitate the creation of large numbers of devices (potentially thousands to millions).
Thus, the present invention seeks to provide a method and apparatus which overcomes the disadvantages associated with the prior art and allows for the measurement of parameters and detection of properties of fluids on a small scale contained in such as microfluidic devices.
Specifically, the present invention provides a system which is based on using an optical cavity to gain an enhancement of, in principle, greater than one hundredfold over conventional absorption spectroscopy. Furthermore, whilst the invention deals with the measurement of absorption parameters in small volumes of liquid, the mechanism for enhancement of sensitivity is based on CEAS and not total internal reflection, unlike many of the methods of the prior art.
Although the presently disclosed system requires the deposition of mirrors on a microfluidic device, the mechanism for increasing the sensitivity of the absorption measurement is entirely different to that which is employed in, for example, the prior art method of U.S. Pat. No. B6,224,830. Thus, in the present CEAS approach, the light source is transmitted through the entrance mirror into the optical cavity, where it is typically undergoes 100 reflections before it is transmitted through the second mirror and onto the detector. The present system thereby provides a higher number of reflections, and the CEAS approach results in the interrogated sample volume being greatly reduced.
In addition, the present CEAS technique facilitates potential pathlength enhancement of a factor of 100, and allows for the integration of CEAS mirrors directly onto a microfluidic device using a microfabrication approach. This approach has typical mirror separations in the micrometre and sub millimetre ranges, allowing for interrogated sample volumes which generally fall in the range of between 1 femtolitre and 25 nanolitres. Interrogated sample volumes frequently fall between 1 and 100 picolitres, for example, and such values are in the region of around 1000-fold lower than is the case with prior art documents such as U.S. Pat. No. B6,839,140, wherein the minimum interrogated sample volume is about 500 nanolitres. Indeed, the flow cell used within the prior art system of U.S. Pat. No. B6,839,140 receives the liquid sample from an external source, such as an analytical separation column attached to HPLC or CE instruments, whereas the present approach allows both CEAS detection and complex fluidic processing, such as analytical separations, to be integrated on the same microfluidic device.